Despite the success of genome projects at listing every gene, there is still a great deal to be understood about how the large majority of genes function in complex biological processes. This is especially true in mammals, where high-throughput genetic approaches are either not feasible or are very time-consuming. RNA interference (RNAi) screens in cell lines have proven extremely useful in this regard;in particular, RNAi and micro-RNA (miRNA) screens that harness the power of biological selection in vivo have enormous potential for the future. In this application, we request funding to build an RNAi/ miRNA screening facility that addresses two of the five research themes of the RFA. (i) The facility will greatly enhance our ability to "apply genomics and other high-throughput technologies" to studies of immune function, by providing the high-throughput instruments and RNAi/ miRNA libraries needed for the large-scale RNAi and miRNA screens outlined below. (ii) As a resource intended to be self-sustaining by the end of the proposed 3-year project period, the facility will "reinvigorate and energise the local biomedical research community" in San Diego by providing instrumentation, reagents and advice on RNAi screen design that we believe will catalyze new discoveries, collaborations and cross-disciplinary research. The facility will be based at the La Jolla Institute for Allergy &Immunology (LIAI) and will be used initially by researchers at the LIAI and The Scripps Research Institute (TSRI) to investigate questions of fundamental importance to immunology. In Project 1 (LIAI), we will use a genome-wide siRNA screen to identify missing players in the cellular response to double-stranded (ds) RNA, and a focused lentiviral shRNA screen to identify the unknown DNA-binding protein(s) that initiate responses to foreign microbial dsDNAs. In Project 2 (TSRI), we will screen a comprehensive library of miRNA-expressing lentiviruses for their ability to abrogate B cell tolerance in vivo. In Project 3 (LIAI), we will use this miRNA library, as well as a focused lentiviral shRNA library directed against transcription factors and chromatin-associated proteins, to identify candidates that influence the decision of CD8 T cells to express markers of effector or memory cell fate. In Project 4 (LIAI), we will use this lentiviral shRNA library to identify transcription factors and chromatin-associated proteins that influence the ability of iNKT cells to express the cytokines interleukin-4 (IL-4) and interferon-3 (IFN-3), first in cultured human cell lines and later in pooled shRNA screens in vivo. Together these studies use state-of-the art approaches -- cell-based screening formats with multiple simultaneous readouts, and sophisticated pooled screening technology that takes advantage of powerful biological selection in vivo -- to extend our understanding of immune function in diverse cellular contexts. PUBLIC HEALTH RELEVANCE: Although all human genes have been identified, the actual functions of most genes are still mysterious. Recently, an experimental technique known as RNAi screening has become very useful to understand how genes function. We are proposing to set up a facility for RNAi screening that would allow scientists to ask fundamental questions relating to how the body fights off infectious and autoimmune diseases and cancers. This important resource would be broadly available to the general scientific community.